The goal of this project is to develop innovative in vivo DC targeting approach for delivery of a novel DRibble vaccine for cancer immunotherapy and generate the proof-of-principle data and rationale for new clinical trials using the next generation DRibble vaccine. The novel DRibble vaccine provides a new source of tumor antigens derived from short-lived proteins packaged within autophagosomes. Preclinical studies demonstrated vaccines based on DRibble ex vivo loading onto DC are significantly more efficacious in tumor-bearing mice than current vaccines based on whole tumor cells. Cross-presentation of antigens in the DRibbles requires the CD8+ DC subsets that are specialized in cross-priming of naive T cells in vivo. Targeting antigens to this subset of DC with antibodies has been proven a more efficient method to deliver antigens to cross-priming CD8+ T cells. These antibodies recognize receptors that are not limited to CD8+ DC subsets and also expressed on surface of cells other than DC. Recently, a novel c-type lectin, Clec9a, was identified and shown to be exclusively expressed by DC and selectively by the CD8+DC subset. Targeting model or tumor associated antigens via antibodies to this receptor was highly efficient to cross-prime CTL. This new receptor opens up an unprecedented opportunity to efficiently target DRibble to pAPC specialized in cross-priming in vivo. In vivo targeting will also remove the significant hurdle of both labor intensive and costly ex vivo manipulation of DC. At the same time, it could potentially increase the efficiency of antigen capture, processing, and presentation by delivering antigens to the putative 'cross-presentation compartment'of DC specialized in cross-priming. Studies proposed in this application are risky (antibody targeting of DRibble has not been done), but potentially highly rewarding (huge improvement in vaccine design and large impact on DC based cancer vaccines). PUBLIC HEALTH RELEVANCE: We have developed a novel cancer vaccine based on the autophagosomes (a specialized cellular compartment that collects cellular waste and damaged organelles) derived from tumor cells. The goal of this application is to design and test the next DRibble vaccines that will target DRibbles to professional antigen presenting cells that specialized in cross-priming of naive T cells. Data generated from this innovative grant will be used to support future clinical trials.